剪切应力环境下的内皮祖细胞对肝星状细胞增殖和生物功能的影响

目的：研究流体剪切应力条件下的内皮祖细胞（EPCs）对肝星状细胞（HSCs）增殖、粘附、迁移、凋亡等生物学功能以及成纤维化因子α-平滑肌肌动蛋白（α-SMA）、胶原I （Col-I）、胶原III （Col-III）表达的影响。方法：将HSCs与EPCs分别接种于共培养小室的上层和下层,共培养24 h后,给EPCs细胞施加12 dyne/cm²剪切应力,持续24 h。消化细胞,采用CCK-8法检测HSCs的增殖;流式细胞术检测HSCs的凋亡率;细胞贴壁法检测HSCs的粘附功能;Boyden小室检测HSCs的迁移;荧光定量PCR法及Western blot分别检测HSCs的α-SMA、Col-I、Col-III mRNA和蛋白质的表达情况。结果：在剪切应力条件下,EPCs生态小境能明显抑制HSCs的增殖、粘附和迁移能力,促进HSCs凋亡,下调HSCs中Col-I、Col-III mRNA和蛋白质的表达。结论：在剪切应力条件下,EPCs生态小境对HSCs纤维化的发展具有一定抑制作用。     

Activation of heme oxygenase-1 by laminar shear stress ameliorates high glucose-induced endothelial cell and smooth muscle cell dysfunction

High glucose (HG)-induced endothelial cell (EC) and smooth muscle cell (SMC) dysfunction is critical in diabetes-associated atherosclerosis. However, the roles of heme oxygenase-1 (HO-1), a stress-response protein, in hemodynamic force-generated shear stress and HG-induced metabolic stress remain unclear. This investigation examined the cellular effects and mechanisms of HO-1 under physiologically high shear stress (HSS) in HG-treated ECs and adjacent SMCs. We found that exposure of human aortic ECs to HSS significantly increased HO-1 expression; however, this upregulation appeared to be independent of adenosine monophosphate-activated protein kinase, a regulator of HO-1. Furthermore, HSS inhibited the expression of HG-induced intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and reactive oxygen species (ROS) production in ECs. In an EC/SMC co-culture, compared with static conditions, subjecting ECs close to SMCs to HSS and HG significantly suppressed SMC proliferation while increasing the expression of physiological contractile phenotype markers, such as α-smooth muscle actin and serum response factor. Moreover, HSS and HG decreased the expression of vimentin, an atherogenic synthetic phenotypic marker, in SMCs. Transfecting ECs with HO-1-specific small interfering (si)RNA reversed HSS inhibition on HG-induced inflammation and ROS production in ECs. Similarly, reversed HSS inhibition on HG-induced proliferation and synthetic phenotype formation were observed in co-cultured SMCs. Our findings provide insights into the mechanisms underlying EC-SMC interplay during HG-induced metabolic stress. Strategies to promote HSS in the vessel wall, such as continuous exercise, or the development of HO-1 analogs and mimics of the HSS effect, could provide an effective approach for preventing and treating diabetes-related atherosclerotic vascular complications.     

Co-culture of endothelial cells and smooth muscle cells affects gene expression of angiogenic factors

Endothelial cells (EC) are in contact with the underlying smooth muscle cells (SMC). The interactions between EC and SMC in the vessel wall are considered to be involved in the control of growth and function of blood vessels. A co-culture system of EC and SMC and a method for separation of these cells was developed in order to investigate whether the presence of physical contact between EC and SMC affected the gene expression of angiogenic factors. Human EC and SMC were prepared from the great saphenous veins. Autologous EC were added on top of the confluent layer of SMC. After 72 h in co-culture, the EC were magnetically separated from SMC with the use of superparamagnetic beads. RT-PCR products for bFGF, bFGFR, VEGF, PDGF-AA, PDGF-BB, TGF-beta, and beta-actin were analyzed to study the mRNA expressions. The protein level of selected factors was studied by ELISA technique. In co-cultured SMC there was a statistically significant higher gene expression of VEGF, PDGF-AA, PDGF-BB, and TGF-beta and significant lower gene expression of bFGF and its receptor than in single cultured SMC. The protein level of PDGF-BB and TGF-beta was also significantly higher in co-cultured SMC. In co-cultured EC there were no significant differences in gene expression of PDGF-AA, PDGF-BB, and TGF-beta compared with single cultured EC. The gene expression and protein synthesis of VEGF was significantly higher in co-cultured EC. The findings from the present study suggest that cell-cell interactions of EC and SMC affect the gene and protein expression of angiogenic factors.     

Research of smooth muscle cells response to fluid flow shear stress by hyaluronic acid micro-pattern on a titanium surface

The morphology of vascular smooth muscle cells (SMCs) in the normal physiological state depends on cytoskeletal distribution and topology beneath, and presents vertical to the direction of blood flow shear stress (FFSS) although SMCs physiologically are not directly exposed to the shear conditions of blood flow. However, this condition is relevant for arteriosclerotic plaques and the sites of a vascular stent, and little of this condition in vitro has been studied and reported till now. It is unclear what will happen to SMC morphology, phenotype and function when the direction of the blood flow changed. In this paper, the distribution of SMCs in a specific area on Ti surface was regulated by micro-strips of hyaluronic acid (HA). Cell morphology depended on the distribution of the cytoskeleton extending along the micrographic direction. Simulated vascular FFSS was perpendicular or parallel to the direction of the cytoskeleton distribution. Based on investigating the morphology, apoptotic number, phenotypes and functional factors of SMCs, it was obtained that SMCs of vertical groups showed more apoptosis, expressed more contractile types and secreted less TGF-β1 factor compared with SMCs of parallel groups, the number of ECs cultured by medium from SMCs of parallel groups was larger than vertical groups. This study could help to understand the effect of direction change of FFSS on patterned SMC morphology, phenotype and function.     

Cytotoxicity and genome-wide microarray analysis of intestinal smooth muscle cells in response to hexavalent chromium induction

Chronic ingestion of high concentrations of hexavalent chromium Cr(VI) in drinking water induces intestinal tumors in mice; however, information on its toxicity on intestinal smooth muscle cells is limited. The present study aimed to assess the in vitro and in vivo toxicological effects of Cr(VI) on intestinal smooth muscle cells. Human intestinal smooth muscle cells (HISM cells) were cultured with different concentrations of Cr(VI) to evaluate effects on cell proliferation ability, oxidative stress levels, and antioxidant system. Furthermore, tissue sections in Cr(VI) exposed rabbits were analyzed to evaluate toxicity on intestinal muscle cells in vivo. Gene chips were utilized to assess differential gene expression profiles at the genome-wide level in 1 μmol/L Cr(VI) treated cells. Intestinal tissue biopsy results showed that Cr(VI) increased the incidences of diffuse epithelial hyperplasia in intestinal jejunum but caused no obvious damage to the structure of the muscularis. Cell proliferation analysis revealed that high concentrations (≥64 μmol/L) but not low concentrations of Cr(VI) (≤16 μmol/L) significantly inhibited the growth of HISM cells. For oxidative stress levels, the expression of reactive oxygen species (ROS) and nitric oxide (NO) was elevated at high concentrations (≥64 μmol/L) but not at low concentrations of Cr(VI) (≤16 μmol/L). In addition, dose-dependent increases in the activity of oxidized glutathione (GSSH)/total-glutathione (T-GSH) were also observed. Gene chip screened 491 differentially expressed genes including genes associated with cell apoptosis, oxidations, and cytoskeletons. Some of these differentially expressed genes may be unique to smooth muscle cells in response to Cr(VI) induction.     

Physiological effects of androgens on human vascular endothelial and smooth muscle cells in culture

Androgenic hormones are associated with atherosclerotic cardiovascular disease, although the underlying cellular and molecular mechanisms remain unclear. This study examines the impact of androgens on the physiology of human vascular endothelial cells (EC) and smooth muscle cells (SMC) in culture. Cells were incubated with testosterone, dihydrotestosterone (DHT) or dehydroepiandrosterone (DHEA) at various physiological concentrations (5-50 nM) in the present or absence of an androgen receptor (AR) blocker flutamide (100 nM). Cell growth and death, DNA and collagen synthesis, and gene protein expression were assessed. It was shown that: (1) DHEA protected EC from superoxide injury via AR-independent mechanisms; (2) testosterone induced DNA synthesis and growth in EC via an AR-independent manner with activation of ERK1/2 activity; (3) DHT inhibited DNA synthesis and growth in EC in an AR-dependent manner; (4) testosterone and DHT enhanced ERK1/2 activation and proliferation in SMC via AR-independent and -dependent pathways, respectively; and (5) these androgens did not significantly affect collagen synthesis in SMC. We conclude that androgens possess multiple effects on vascular cells via either AR-dependent or -independent mechanisms. Testosterone and DHEA may be “beneficial” in preventing atherosclerosis by improving EC growth and survival; in contrast, stimulation of VSMC proliferation by testosterone and DHT is potentially “harmful”. The relationship of these in vitro effects by androgens to in vivo vascular function and atherogenesis needs to be further clarified.     

豚鼠耳蜗螺旋动脉平滑肌细胞和内皮细胞静息膜电位特性

目的:多种内耳疾病和内耳微循环障碍有关,但目前对提供内耳主要血供的耳蜗螺旋动脉平滑肌(SMC)和内皮细胞(EC)的生理学特性还不十分清楚,需要进一步研究。方法:本研究采用双细胞内微电极记录技术和细胞荧光染色技术,研究耳蜗螺旋动脉平滑肌和内皮细胞的膜电位特性和细胞间的通讯联系。结果:研究发现耳蜗螺旋动脉SMC和EC具有高、低两种静息膜电位(RP)状态,两种静息膜电位状态的细胞对乙酰胆碱和高K+的反应完全不同。双微电极可同时记录到EC-ECS、MC-SMC和SMC-EC不同类型的细胞,两个细胞的静息膜电位也可以是双高RP、双低RP和一高一低RP。实验所记录的一高一低RP均是SMC-EC类型,而且EC初始膜电位均为高电位,SMC初始膜电位均为低电位。而双高RP和双低RP可以是SMC-SMC或EC-EC或SMC-EC类型。结论:结果表明耳蜗螺旋动脉的SMC和EC在0.3~0.5 mm的范围内,同类细胞之间有很好的通讯联系,能很好的保持功能的协同和一致,血管壁异类细胞则不同。     

Comparative endocrinology-paracrinology-autocrinology of human adult large vessel endothelial and smooth muscle cells

Summary Endothelial and smooth muscle cells were isolated from human adult large blood vessels to compare their proliferative response to hormones and growth factors. Neural extracts and the medium from differentiated hepatoma cells were used as concentrated sources of required hormones and growth factors that supported both cell types. Active hormones and growth factors were identified from the neural extracts and hepatoma medium by substitution or direct isolation and biochemical characterization. Epidermal growth factor, lipoproteins, and heparin-binding growth factors elicited growth-stimulatory effects on both endothelial and smooth muscle cells. Both types of human vascular cells displayed 7600 to 8600 specific heparin-binding growth factor receptors per cell with a similar apparent dissociation constant (Kd) of 200 to 250 pM. Heparin modified the response of both endothelial and smooth muscle cells to heparin-binding growth factors dependent on the type of heparin-binding growth factor and amount of heparinlike material present. In addition, heparin exerted a growth factor-independent inhibition of smooth muscle cell proliferation. Platelet-derived growth factor, insulinlike growth factors, and glucocorticoid specifically supported proliferation of smooth muscle cells with no apparent effect on endothelial cell proliferation. Growth-factorlike proteinase inhibitors had an impact specifically on endothelial cell proliferation. Transforming growth factor beta was a specific inhibitor of endothelial cells, but had a positive effect on smooth muscle cell proliferation. The results provide a framework for differential control of the two vascular cell types at normal and atherosclerotic blood vessel sites by the balance among positive and negative effectors of endocrine, paracrine and autocrine origin. This research was supported by NIH grants CA37589, HL33847, and AM35310 from the National Institutes of Health, Bethesda, MD; grant 1718 from the Council for Tobacco Research; and a grant from RJR/Nabisco, Inc.     

Synergistic effect of shear stress and streptavidin-biotin on the expression of endothelial vasodilator and cytoskeleton genes

Dual ligand treatment of streptavidin(SA)-biotin and fibronectin (Fn) enhances the adhesion of endothelial cells (EC) onto synthetic surfaces and promotes the quiescent phenotype of adherent EC. The current study investigates the effect of the dual ligand on the expression of endothelial genes in static culture and under shear stress (4 h at 10 dynes/cm2). Expression of 23 genes in the classes of signaling, cytoskeleton/ECM, vasoregulation, and shear-responsive were examined. Eight genes (argininosuccinate synthetase, K+ channel, TGFbeta, Mn-SOD, alpha-tubulin, t-PA, COX2, and eNOS) were significantly upregulated by shear stress. Two genes (caveolin-1 and ET-1) were downregulated by shear stress. Three genes (RhoA, elastin, alpha-actinin) were upregulated by the dual ligand treatment in static culture, and four genes (FAK, elastin, COX2, and eNOS) were upregulated when the dual ligand and shear stress were applied simultaneously. Northern blot analyses on FAK, RhoA, elastin, and alpha-actinin revealed similar results. The results suggest (1) the use of SA-biotin to supplement EC adhesion enhances the integrity of the EC cytoskeleton by upregulating the expression of cytoskeleton/ECM genes, and (2) a likely relationship between the expression of cytoskeleton/ECM genes and the downstream events, such as the shear-induced expression of eNOS and COX2 genes. Analyses presented in this study provide insights into the mechanism by which SA-biotin-supplemented EC mediate gene expression.     

Porcine aortic endothelial cells show little effects on smooth muscle cells but are potent stimulators of cardiomyocyte growth

Smooth muscle cells (SMC) and endothelial cells (EC) play a pivotal role in arteriogenesis and atherosclerosis. We evaluated the role of EC on the growth of SMC and neonatal cardiomyocytes (NEO) by using serum-free EC-supernatant (AoCM). Five percent fetal calf serum was used in order to mimic growth effects of blood. EC and SMC purities were 99% as determined by absence or presence of markers such as CD31, desmin, -smooth muscle actin and tropomyosin using immunostaining and FACS analysis. AoCM markedly influenced the morphology of NEO as determined by -actinin staining but showed only little effect on the phenotype of SMC. Protein synthesis after 2 days increased 2.5-fold in SMC and 3.7-fold in NEO as determined by tritium incorporation. The values for serum (2.8 and 2.3-fold, respectively) were comparable. The induction of DNA-synthesis by serum in NEO was twice that of AoCM (3.9-fold). However, proliferative effects of serum and AoCM on SMC differed markedly: Serum induced a 66-fold increase in DNA-synthesis resulting in a 54% higher cell number. DNA-synthesis after AoCM treatment lead to a nonsignificant small increase and no proliferation was detected. Platelet derived growth factor (PDGF-AB), present in blood, induced a 47-fold increase in DNA-synthesis and a 38% increase in cell number. Our data suggest that EC in the absence of physical forces exert strong morphogenic effects on cardiomyocytes but they lack specific effects on smooth muscle cells. In vessels EC might function as a border to isolate SMC from key regulators in blood such as PDGFs.     

Epigenetic response of endothelial cells to different wall shear stress magnitudes: A report of new mechano-miRNAs

Endothelial cells (ECs) respond to flow stress via a variety of mechanisms, leading to various intracellular responses that can modulate the vessel wall and lead to diseases if the flow is disturbed. Mechano-microRNAs (miRNAs) are a subset of miRNAs in the ECs that are flow responsive. Mechano-miRNAs were shown to be related to atherosclerosis pathophysiology, and a number of them were identified as pathologic. Here, we exposed human carotid ECs to different wall shear stresses (WSS), high and low, and evaluated the response of miRNAs by microarray and quantitative polymerase chain reaction analysis. We discovered five new mechano-miRNAs that were not reported in that context previously to the best of our knowledge. Moreover, functional pathway analysis revealed that under low WSS conditions, several pathways regulating apoptosis are affected. In addition, KLF2 and KLF4, known atheroprotective genes, were downregulated under low WSS and upregulated under high WSS. KLF2 and VCAM1, both angiogenic, were upregulated under high WSS. NOS3, which is vascular protective, was also upregulated with higher WSS. On the contrary, ICAM-1 and E-selectin, both atherogenic and proinflammatory, were upregulated with high WSS. Collectively, the epigenetic landscape with the gene expression analysis reveals that low WSS is associated with a proapoptotic state, while high WSS is associated with a proliferative and proinflammatory state.     

Cytoplasmic calcium response to fluid shear stress in cultured vascular endothelial cells

Summary Vascular endothelial cells modulate their structure and functions in response to changes in hemodynamic forces such as fluid shear stress. We have studied how endothelial cells perceive the shearing force generated by blood flow and the substance(s) that may mediate such a response. We identify cytoplasmic-free calcium ion (Ca⁺⁺), a major component of an internal signaling system, as a mediator of the cellular response to fluid shear stress. Cultured monolayers of bovine aortic endothelial cells loaded with the highly fluorescent Ca⁺⁺-sensitive dye Fura 2 were exposed to different levels of fluid shear stress in a specially designed flow chamber, and simultaneous changes in fluorescence intensity, reflecting the intracellular-free calcium concentration ([Ca⁺⁺] _i ), were monitored by photometric fluorescence microscopy. Application of shear stress to cells by fluid perfusion led to an immediate severalfold increase in fluorescence within 1 min, followed by a rapid decline for about 5 min, and finally a plateau somewhat higher than control levels during the entire period of the stress application. Repeated application of the stress induced similar peak and plateau levels of [Ca⁺⁺] _i but at reduced magnitudes of response. These responses were observed even in Ca⁺⁺-free medium. Thus, a shear stress transducer might exist in endothelial cells, which perceives the shearing force on the membrane as a stimulus and mediates the signal to increase cytosolic free Ca⁺⁺. This work was partly supported by a grant-in-aid, for Special Project Research no. 61132008, from the Japanese Ministry of Education, Science and Culture and a research fund from the Atherosclerosis Study Association.     

Effect of spatial gradient in fluid shear stress on morphological changes in endothelial cells in response to flow

Arterial bifurcations are common sites for development of cerebral aneurysms. Although this localization of aneurysms suggests that high shear stress (SS) and high spatial SS gradient (SSG) occurring at the bifurcations may be crucial factors for endothelial dysfunction involved in aneurysm formation, the details of the relationship between the hemodynamic environment and endothelial cell (EC) responses remain unclear. In the present study, we sought morphological responses of ECs under high-SS and high-SSG conditions using a T-shaped flow chamber. Confluent ECs were exposed to SS of 2-10 Pa with SSG of up to 34 Pa/mm for 24 and 72 h. ECs exposed to SS without spatial gradient elongated and oriented to the direction of flow at 72 h through different processes depending on the magnitude of SS. In contrast, cells did not exhibit preferred orientation and elongation under the combination of SS and SSG. Unlike cells aligned to the flow by exposure to only SS, development of actin stress fibers was not observed in ECs exposed to SS with SSG. These results indicate that SSG suppresses morphological changes of ECs in response to flow.     

兔主动脉内皮细胞和平滑肌细胞共培养体系的建立

目的：建立兔主动脉内皮细胞（ECs）和平滑肌细胞（SMCs）共培养体系,为进一步开展相关研究打下基础。方法：以Transwell膜为载体,将原代培养的ECs接种在Transwell膜的一侧,将SMCs接种于膜的另一侧或培养板底部,模拟血管壁的结构关系,建立两种共培养体系,并利用电镜对其进行观察。结果：原代培养的ECsⅧ因子免疫细胞化学染色阳性,ECs和SMCs在Transwell膜上生长良好,ECs单层生长,呈“鹅卵石”样外观;SMCs多层生长,呈明显“峰-谷”状外观。膜两侧的ECs和SMCs可以发生细胞连接。结论：我们建立的兔ECs和SMCs共培养体系是成功的,能较好地模拟血管壁的结构关系。     

Characterization of smooth muscle differentiation of purified human skeletal muscle‐derived cells

The purpose of this study is to characterize the smooth muscle differentiation of purified human muscle‐derived cells (hMDCs). The isolation and purification of hMDCs were conducted by modified preplate technique and Dynal CD34 cell selection. Smooth muscle cell differentiation was induced by the use of smooth muscle induction medium (SMIM) and low‐serum medium. The gene expressions at the mRNA and protein levels of undifferentiated and differentiated hMDCs were tested by RT‐PCR, Western blot and immunofluorescence studies. Western blot and immunofluorescence studies demonstrated the purified hMDCs cultured in SMIM for 4 weeks and expressed significant amount of smooth muscle myosin heavy chain (MHC) and α‐smooth muscle actin (ASMA). The cells cultured in low‐serum medium for 4 weeks also expressed ASMA, while the control group did not. RT‐PCR analysis showed increased gene expression of smooth muscle markers, such as ASMA, Calponin, SM22, Caldesmon, Smoothelin and MHC when purified hMDCs were exposed to SMIM for 2 and 4 weeks when compared to the controls. In conclusion, we confirmed the smooth muscle differentiation capability of purified hMDCs. The gene expression of smooth muscle differentiation of purified hMDCs was characterized. These cells may be potential biomaterials for human tissue regeneration.     

Growth inhibitory effects of dimethyl sulfoxide and dimethyl sulfone on vascular smooth muscle and endothelial cells in vitro

Summary The growth of bovine aortic smooth muscle and endothelial cells was studied after exposure to dimethyl sulfoxide (DMSO) or its major metabolite, dimethyl sulfone (DMSO₂). Both compounds caused a dose-dependent inhibition of cell growth as determined by [³H]thymidine incorporation and by counting the number of cells with time of exposure in culture. The IC₅₀ of DMSO (concentration which produces 50% inhibition of growth) was 1% for smooth muscle cells and 2.9% for endothelial cells. Similarly, the IC₅₀ of DMSO₂ was also 1% for smooth muscle cells, but was 1.8% for endothelial cells. After a 4-d exposure to either compound, the growth inhibition of smooth muscle cells was completely reversible at 1%, partially reversible at 2 to 3% and completely irreversible at 4%. By comparison, inhibition of endothelial cell growth was completely reversible up to 4% of either compound. It is concluded that the growth of smooth muscle cells was similarly inhibited by DMSO, and DMSO₂, but that smooth muscle cells were more susceptible than endothelial cells to the growth inhibitory effects of these compounds. In addition, DMSO₂ was a more potent inhibitor of cell growth than DMSO and its growth inhibition was less reversible than that produced by DMSO.     

Cyclic strain induces expression of specific smooth muscle cell markers in human endothelial cells

The objective of this study was to determine whether cyclic strain could promote human umbilical vein endothelial cells (HUVECs) to express markers in common with the mature smooth muscle cell (SMC) phenotype, suggesting endothelial cell to SMC transdifferentiation. HUVECs were cultured on stretched membranes at 10% stretch and 60 cycles/min for 24-96 hr, and demonstrated elongation with enhanced and organized F-actin distribution. By using real-time polymerase chain reaction analysis, the mRNA levels of five specific SMC markers, SM22-alpha, alpha-smooth muscle actin (alpha-SMA), caldesmon-1, smooth muscle myosin heavy chain (SMMHC), and calponin-1 were significantly increased in cyclic strain-treated HUVECs as compared with those in static control cells. Protein levels of SM22-alpha and alpha-SMA were also substantially increased by Western blot and immunofluorescence staining. In addition, two specific endothelial markers, von Willebrand factor (vWF) and vascular endothelial growth factor receptor-2 (VEGFR-2), showed a reduction in mRNA expression. In addition, cyclic strain-induced increase of SM22-alpha and alpha-SMA expression were reversible when cells were cultured back to the static condition. These results demonstrate a possible endothelial cell to SMC transdifferentiation in response to cyclic strain. Hemodynamic forces in modulating endothelial phenotype may play an important role in the vascular system.